Peptidoglycan (PG; murein) hydrolases play diverse roles in the biology and pathogenesis of Streptococcus pneumoniae (pneumococcus) and other human bacterial pathogens. Pneumococcal PG hydrolases that mediate stress-induced autolysis and fratricide during competence have been studied for some time. In contrast, little is known about the membrane-associated PG hydrolases that remodel PG during cell division and are required for normal growth, cell shape, chaining, and virulence of S. pneumoniae. The activities of division PG hydrolases have to be carefully regulated to coordinate PG cleavage with stage of cell division and thereby prevent catastrophic damage to cells. The overall goal of this proposal is to fill in this major knowledge gap by determining the functions, regulatory interactions, and mechanisms of integration with cell division of the FtsEX:PcsB and Pmp23 (PvaA) PG hydrolases of S. pneumoniae. These two PG hydrolases will be used as models to test the two central hypotheses of this proposal: (i) PG hydrolases involved in cell division are autoinhibited, and their activation is strictly controlled by interactions with regulatory proteins that relieve autoinhibition at precise times during cell division. This hypothesis is most developed for the FtsEX:PcsB PG hydrolase from previous work and will be extended to Pmp23, which is an important division protein that likely functions as a lytic transglycosylase. (i) PG cleavage by division PG hydrolases allows the entry and synthesis of new glycan strands by the PG synthesis machinery and also leaves signals in the PG for other enzymes that metabolize PG. A block in PG synthesis would account for a characteristic rounded cell shape that occurs upon depletion of PcsB or Pmp23. Two specific aims will be accomplished to meet the overall goal of this proposal. Aim 1 will determine the functions and signal transduction, assembly and interactions with other proteins, and roles in PG biosynthesis of the FtsEX:PcsB complex during pneumococcal cell division. Aim 2 will determine the functions and biochemical activity, roles in PG biosynthesis, and localization and interactions with other proteins of Pmp23 during pneumococcal cell division. These aims will be accomplished by a powerful, comprehensive strategy that combines bacterial genetics, physiology, cell biology, enzymology, and structural biology. Results from this proposal will make a major contribution to understanding the roles and regulation of these two important division PG hydrolases in S. pneumoniae and will have wide-spread impact on the fields of bacterial PG biosynthesis and cell division, especially of prolate-ellipsoid shaped ovococcus species, which include many major pathogens. The FtsX protein is a likely target for bactericidal chemokines, and results from this proposal will provide information for understanding the mechanism of this innate-immune killing. Work in this proposal will provide validation and further development of methods of bacterial genetics and cell biology of ovococcus bacteria. Finally, this proposal has the potential to provide new bacterial surface targets for antibiotic and vaccine development.